Accumulation and penetration behavior of hypericin in glioma tumor spheroids studied by fluorescence microscopy and confocal fluorescence lifetime imaging microscopy

Abstract

Glioblastoma WHO IV belongs to a group of brain tumors that are still incurable. A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell-cell interactions and the extracellular matrix is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O₂ activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time > 30 min is required to achieve most suitable conditions for an effective PDT.

Keywords: Fluorescence lifetime; Fluorescence microscopy; Hypericin; Photodynamic therapy; Tumor spheroid.

Fig. 1

a Schematic illustration of the tumor spheroid formation in a 96-well plate by the forced-floating method. Due to a cell-repellent coating inside the well, the included cell suspension is forced to agglomerate to a spheroid. Additionally, the round bottom of the cavities promotes and accelerates the formation of spherical tumor clusters in reproducible size and shape. b Phase contrast image of a U-87 MG tumor spheroid with a seeding density of 1500 cells/cm² and a cultivation time of 35 h + 15 min

Fig. 2

Investigation of hypericin penetration into spheroids by comparing outer and inner spheroid sections. The penetration was examined with fluorescence microscopy and FLIM for an incubation time of 5 min. Fluorescence images of the whole spheroid (a) and spheroid sections (b, c) are displayed. The fluorescence intensity of Hoechst in the nuclei (blue) and hypericin fluorescence (red) is superimposed in a–c. The outer section reveals a homogeneous hypericin intensity (red) throughout the section compared to the inner one (b, c). In the inner section, an intensity gradient is observed (c). All fluorescence images have scale bars of 25 µm. To represent FLTs across the sections, FLIM images (24 × 24 µm) are superimposed on corresponding brightfield images to generate a composite image (d, e). Scale bars of the brightfield images represent 25 µm. Hypericin FLTs vary between 3.5 and 2.5 ns depending on the position inside the spheroid (peripheral to central area). An FLT gradient can be observed from the spheroid outside (3.5 ns) to the inside (2.5 ns) (e)

Fig. 3

Influence of different incubation concentrations on the hypericin uptake and penetration, investigated by fluorescence microscopy. Fluorescence images of whole spheroids, incubated with different hypericin concentrations (0.05 µM, 0.5 µM, 1.25 µM, 2.5 µM) for 30 min, are illustrated in a, c, e, and g. Corresponding fluorescence images of their inner sections are displayed in b, d, f, and h. The Hoechst fluorescence intensity in the nuclei (blue) proves cell occurrence throughout the whole spheroids. Hypericin fluorescence intensity (red) of whole spheroids increases with larger incubation concentrations (a, c, e, g). As shown by the inner sections, an increase in incubation concentration also results in a higher internal intensity, at least partly, in an annular area at the section edges (b, d, f, h; red). Intensity gradients occur for all investigated concentrations (b, d, f, h), although hardly visible for the 2.5 µM incubation concentration (h). The scale bars of all fluorescence images are 25 µm

Fig. 4

Impact of different incubation times on hypericin at the spheroid center, examined by fluorescence microscopy and FLIM. Inner sections of the spheroids were investigated by both techniques after incubation times of 5 min, 30 min, 125 min, 10 h + 30 min, and 35 h + 15 min, respectively. Cell nuclei were identified by their blue Hoechst fluorescence (a–e). Fluorescence images of the sections reveal hypericin intensity gradients (red) for a 5 min, 30 min, and 125 min incubation, with decreasing intensity towards the core (a–c). An evenly distributed hypericin fluorescence intensity is observed for long incubation times of 10 h + 30 min and 35 h + 15 min (d, e). Additionally, a larger amount of hypericin penetrates deeper into the spheroid at longer incubation times. Overall, an increasing hypericin fluorescence intensity (red) is observed for increasing incubation times (a–e). The scale bars of all fluorescence images are 25 µm. Composite images were again generated by combining FLIM images (24 × 24 µm) and corresponding brightfield images (f–j). Corresponding scale bars of the brightfield images also equal 25 µm (f–j). Composite FLIM images show an FLT range of 2.5–4.5 ns for hypericin throughout the inner sections of the tumor spheroids (f–j). Gradients of hypericin FLT appear for short incubation times (f, g), whereas a mostly homogeneous FLT distribution occurs at longer incubation times (h–j)

Fig. 5

Hypericin fluorescence spectra of inner spheroid sections treated for different incubation times (a) and FLTs as well as hypericin maxima intensities depending on pH (b). In a, fluorescence spectra are averaged line scans across the sections and normalized to maximum intensity. In b, FLTs of hypericin and maximum intensities of the 1st hypericin fluorescence maximum (600 nm) are shown for different pH levels (pH 4–9). The largest hypericin FLT of 5.9 ns appears at pH 8, whereas the lowest FLT of 4.5 ns is observed at pH 4. In comparison to FLTs, the highest intensity was measured at pH 7 and the lowest at pH 9